Effect of early diet and virus infections on immune regulation and the development islet autoimmunity -Trial to Reduce IDDM in the Genetically at Risk (TRIGR). The study will yield important new information about the main environmental candidate risk factors in the pathogenesis of type 1 diabetes (T1D) utilizing the setup of TRIGR (dietary intervention). The aim is to evaluate associations between n-3 fatty acids, vitamin D, cow's milk exposure and viral infections with indicators of immune regulation and inflammation as well as induction of islet autoimmunity. Islet autoimmunity is defined as repeated positivity fo at least two diabetes-associated autoantibodies out of four ones measured. The current study will be based on biosamples collected in the TRIGR cohort. TRIGR study is an international double-blind randomized clinical trial of 2159 infants with HLA-conferred disease susceptibility and a first-degree relative with T1D recruited between 2002-2007 in 15 countries. In TRIGR an extensively hydrolyzed casein formula is compared to regular cow's milk based one. All subjects are followed until the youngest child will be 10-year-old. Blood samples are collected at 3 to 12 months' intervals. We measure from serum in cohort or nested case-control design: fatty acid composition with gas chromatography; 25-OH-vitamin D concentration with chemiluminescent mircoparticle immunoassay; virus antibodies with plaque neutralization and enzyme immune assay; cow's milk antibodies with enzyme-linked immunosorbent assay; antibodies to dietary bovine insulin by using the competitive radio-immunoassay with radioactive and cold insulin; and cytokines and chemokines with the Milliplex MAP kit. When studying cell-mediated responses to Coxsackievirus B, -lactoglobulin and bovine insulin, we use frozen peripheral blood mononuclear cells (PBMC), from which we measure Treg Th1, Th2 and Th17 markers, FoxP3, CTLA-4, IFN-g, IL-4, IL-5, IL-13, IL-17 and IL-22, using RT-qPCR methodology in cow's milk protein stimulated PBMCs. We will also analyze from frozen PBMCs the epigenetic modulation of two genes (FOXP3, IL-2) responsible for the development of regulatory T-cells. The methods are very well established and well-functioning in our laboratories. The analysis can start straight after the samples are received from TRIGR. This application covers the costs for the analysis of serum 25-OH-vitamin D concentration, serum fatty acids, immune regulation, inflammatory markers, and virus infections. It also covers coordination, data collection, management and statistical analysis as well as sample collection and aliquation for the current study.